Alternative sigma factors play an important role in the regulation of bacterial gene expression. The proposed research will explore the role of an alternative sigma factor, sigmaE, in the regulation of actinomycin biosynthesis in the Gram-positive bacterium, Streptomyces antibioticus. Specific experiments to be performed will include: (1)identification of an RNA polymerase holoenzyme in Streptomyces antibioticus capable of transcribing the phenoxazinone synthase gene (phsA) and verification of sigmaE as the sigma factor associated with that holoenzyme; (2)characterization of the cloned gene for sigmaE from S. antibioticus; (3)examination of the role of sigmaE in the regulation of the expression of phsA and other genes involved in the biosynthesis of actinomycin - Disruption of the sigE gene; (4)expression of the cloned sigE gene in S. antibioticus sigE null mutants; (5)examination of the regulation of sigE expression in S. antibioticus; (6)examination of the role of guanosine tetraphosphate in regulating the expression of the sigE gene; and (7)identification of other genes in S. antibioticus whose transcription is dependent on sigE. The proposed studies will mesh well with the longer term research objectives of the applicant laboratory. In particular, the proposed research will connect directly to ongoing experiments related to the role of highly phosphorylated guanine nucleotides in the regulation of actinomycin biosynthesis and to experiments dealing with the regulation of phenoxazinone synthase expression by glucose. The suggested experiments will add significantly to our understanding of mechanisms involved in the regulation of prokaryotic gene expression generally and to our specific comprehension of mechanisms regulating antibiotic production. As over 70 % of the antibioticus used in clinical and veterinary medicine are produced by members of the genus Streptomyces, a thorough understanding of genetic regulatory mechanisms involved in antibiotic production is essential if these organisms are to be manipulated to produce new and more effective chemotherapeutic agents.